靶向TTF-1的siRNA腺相关病毒载体组装验证

Package and Verification of Adeno-Associated Virus Vector Constructed with siRNA Targeting TTF-1

为了解决靶向TTF-1的siRNA导入肺腺癌细胞的问题,设计了3条靶向NCI-H1975细胞TTF-1基因的siRNA,并将其分别构建到腺相关病毒上.在293细胞中装配病毒,收获病毒原初液后进行超滤浓缩、层析柱纯化,测定病毒滴度.重组病毒感染肺腺癌细胞系NCI-H1975,通过Western-Blot实验检测siRNA效果,利用凋亡实验验证细胞生物学效应.Western-Blot实验测得siRNA有较好的干扰效果,凋亡实验测得肺腺癌细胞NCI-H1975产生了凋亡,从而证明具有感染性的靶向TTF-1的siRNA腺相关病毒载体组装成功.

In order to introduce siRNA targeting TTF-1 gene into lung adenocarcinoma cells,first,three TTF-1-gene-specific siRNAs for NCI-H1975 cells were respectively designed and constructed in adeno-assosiated virus（AAV）.Next,the primary culture liquid of AAV was harvested after the package of AAV in 293 cells.Then,the titer of AAV was detected after the concentration by ultrafiltration and after the purification by column chromatography for the primary culture liquid of AAV.Finally,NCI-H1975 cells were infected by recombinant AAV,and the interference effect of siRNA and the cell biology effect were respectively verified by Western-Blot experiments and cells apoptosis experiments,with good interference effect of siRNA and apoptosis of NCI-H1975 being both revealed,which means that infective AAV-TTF-1-siRNA was successfully constructed.